REGULATION DE L'ACTIVITE ET DE LA LOCALISATION DES PHOSPHATASES CDC25B

Ordered activation of cyclin-dependent kinases (CDK), associated to their regulatory cyclin subunit, controls the progression of eukaryotic cells within the cell cycle. The activity of CDK/cyclin is notably regulated by a balance between inhibitory phosphorylation (Wee, Myt) and activating dephosphorylation by CDC25 phosphatases. In human cells, three dual specificity phosphatases, CDC25A, B and C, are involved in the regulation of these complexes at various stages of the cell cycle. CDC25A is active in G1/S transition while CDC25C controls entry into mitosis. In contrast, CDC25B would operate in S phase as well as at the G2/M transition. The existence of three versions of CDC25B (B1, B2 and B3) resulting from alternative splicing might explain this controversy. In order to study the functions and involvements of each variant of CDC25B, we first studied their regulation by kinase CK2, a kinase which may play a role in G2/M transition. Our in vitro studies indicated that CK2 phosphorylates the three variants of CDC25B, but not CDC25C. Mass spectrometry analysis of CDC25B indicated that at least two residues, serine 186 and 187, are phosphorylated by CK2 in vitro. In addition, CDC25B interacts with CK2 in vivo and in vitro in human and insect cells. Finally, phosphorylation of CDC25B by CK2 increases its phosphatase activity both in vitro and in vivo. Thus CK2 is a positive regulator of the catalytic activity of CDC25B. In the course of the cell cycle or during checkpoint response, CDC25B is also regulated at the level of intracellular location. Indeed, the phosphatase shuttles between cytoplasm and nucleus, and the shuttle may be regulated notably by phosphorylation. We have shown that protein kinase AKT/PKB phosphorylates CDC25B in vitro on serine 353 and leads to cytoplasmic storage. Activation of AKT/PKB by hydrogen peroxide induces the cytoplasmic localization of CDC25B. Mutating serine 353 abolishes CDC25B phosphorylation by ATK/PKB but causes only a delay in cytoplasmic stockpiling of CDC25B, suggesting that other mechanisms participate to this phenomenon. In summary, our studies allowed the identification of two new regulators of CDC25B, CK2 which regulates its catalytic activity, and AKT/PKB which participates to the control of its intracellular location, and have provided a better understanding of the regulation of this phosphatase, even though many other partners still await identification.L'activation séquentielle des Kinases Dépendantes des Cyclines (CDK), associées à leur sous-unité régulatrice la cycline, contrôle la progression des cellules eucaryotes dans le cycle cellulaire. L'activité des complexes CDK/cycline est notamment régulée par une balance entre phosphorylation inhibitrice (Wee, Myt) et déphosphorylation activatrice par les phosphatases CDC25. Dans les cellules humaines, trois phosphatases à double spécificité CDC25A, B et C sont impliquées dans la régulation de ces complexes en différents points du cycle cellulaire. CDC25A agit à la transition G1/S alors que CDC25C contrôle l'entrée en mitose. Par contre, CDC25B agirait en phase S ainsi qu'à la transition G2/M. L'existence de trois variants de CDC25B (B1, B2 et B3) issus d'un épissage alternatif pourrait expliquer cette controverse. Afin d'étudier les rôles et les implications de chaque variant de CDC25B, nous avons d'abord étudié leur régulation par la protéine kinase CK2, kinase qui pourrait jouer un rôle dans le contrôle de la transition G2/M. Nos études in vitro ont démontré que CK2 phosphoryle les trois variants de CDC25B mais pas la protéine CDC25C. Une analyse par spectrométrie de masse de CDC25B indique qu'au moins deux résidus, les sérines 186 et 187, sont phosphorylés in vitro par CK2. De plus, CDC25B interagit avec CK2 in vitro et in vivo dans des cellules humaines et d'insectes. Enfin, la phosphorylation de CDC25B par CK2 augmente son activité phosphatase in vitro ainsi qu'in vivo. CK2 est donc un régulateur positif de l'activité catalytique des CDC25B. Au cours du cycle cellulaire ou en réponse aux points de contrôle, CDC25B est également régulée au niveau de sa localisation intracellulaire. En effet, la phosphatase réalise une navette entre le cytoplasme et le noyau, navette qui peut être régulée notamment par phosphorylation. Nous avons montré que la protéine kinase AKT/PKB phosphoryle in vitro CDC25B sur la sérine 353 et qu'elle provoque son accumulation dans le cytoplasme. L'activation d'AKT/PKB par le peroxyde d'hydrogène reproduit la relocalisation de CDC25B. Par contre, si la mutation de la sérine 353 abolit sa phosphorylation par AKT/PKB, elle n'induit qu'un retard dans la relocalisation cytoplasmique de CDC25B ce qui indique que d'autres mécanismes participent à ce phénomène. Ainsi nos différents travaux ont permis d'identifier deux nouveaux régulateurs de CDC25B, CK2 qui régule son activité catalytique et AKT/PKB qui participe au contrôle de sa localisation intracellulaire, et nous permettent de mieux comprendre la régulation de cette phosphatase même si de nombreux partenaires doivent encore être identifiés

Régulation de l'activité et de la localisation des phosphatases CD25B

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Protein kinase CK2 and cell polarity.

International audienceThere is increasing evidence that protein kinase CK2 is involved, among a wide variety of cellular processes, in the maintenance of mammalian cell morphology and cell polarity. Here, we show that in epithelial cells, a fraction of CK2 is associated to the plasma membrane and that this localization is controlled by cell-matrix interactions. In addition, inhibition of CK2 activity in mammary epithelial cells (MCF10A), using either the specific CK2 inhibitor TBB or siRNA-mediated CK2beta knockdown, induced differential phenotypes revealing an important role of this enzyme in epithelial cell morphology

Highlighting protein kinase CK2 movement in living cells.

International audienceProtein kinase CK2 has traditionally been described as a stable heterotetrameric complex (alpha2beta2) but new approaches that effectively capture the dynamic behavior of proteins, are bringing a new picture of this complex into focus. To track the spatio-temporal dynamics of CK2 in living cells, we fused its catalytic alpha and regulatory beta subunits with GFP and analog proteins. Beside the mostly nuclear localization of both subunits, and the identification of specific domains on each subunit that triggers their localization, the most significant finding was that the association of both CK2 subunits in a stable tetrameric holoenzyme eliminates their nuclear import (Mol Cell Biol 23: 975-987, 2003). Molecular movements of both subunits in the cytoplasm and in the nucleus were analyzed using different new and updated fluorescence imaging methods such as: fluorescence recovery after photo bleaching (FRAP), fluorescence loss in photo bleaching (FLIP), fluorescence correlation spectroscopy (FCS), and photoactivation using a biphoton microscope. These fluorescence-imaging techniques provide unprecedented ways to visualize and quantify the mobility of each individual CK2 subunit with high spatial and temporal resolution. Visualization of CK2 heterotetrameric complex formation could also be recorded using the fluorescence resonance energy transfer (FRET) technique. FRET imaging revealed that the assembling of this molecular complex can take place both in the cytoplasmic and nuclear compartments. The spatio-temporal organization of individual CK2 subunits and their dynamic behavior remain now to be correlated with the functioning of this kinase in the complex environment of the cell

L'Odissea di Petrarca e gli scoli di Leonzio

International audienc

PKB/Akt phosphorylates the CDC25B phosphatase and regulates its intracellular localisation

International audienc

Protein kinase CK2 regulates CDC25B phosphatase activity

International audienc

Phosphorylation of CDC25B by Aurora-A at the centrosome contributes to the G2-M transition.

Aurora-A protein kinase, which is the product of an oncogene, is required for the assembly of a functional mitotic apparatus and the regulation of cell ploidy. Overexpression of Aurora-A in tumour cells has been correlated with cancer susceptibility and poor prognosis. Aurora-A activity is required for the recruitment of CDK1-cyclin B1 to the centrosome prior to its activation and the commitment of the cell to mitosis. In this report, we demonstrate that the CDC25B phosphatase, an activator of cyclin dependent kinases at mitosis, is phosphorylated both in vitro and in vivo by Aurora-A on serine 353 and that this phosphorylated form of CDC25B is located at the centrosome during mitosis. Knockdown experiments by RNAi confirm that the centrosome phosphorylation of CDC25B on S353 depends on Aurora-A kinase. Microinjection of antibodies against phosphorylated S353 results in a mitotic delay whilst overexpression of a S353 phosphomimetic mutant enhances the mitotic inducing effect of CDC25B. Our results demonstrate that Aurora-A phosphorylates CDC25B in vivo at the centrosome during mitosis. This phosphorylation might locally participate in the control of the onset of mitosis. These findings re-emphasise the role of the centrosome as a functional integrator of the pathways contributing to the triggering of mitosis